Long Non-Coding RNA Mir17hg Positively Regulates Melanogenesis by Inhibiting TGFß Receptor 2 under Psychological Stress.

Vitiligo is a common skin depigmentation disorder characterized by the patchy loss of skin color. Nowadays, it is recognized as being correlated with multiple genetic factors as well as the psychological conditions of individuals. Long noncoding RNAs have been reported to underlie the pathogenesis of vitiligo; however, the role of long noncoding RNAs in the stress-related depigmentation process remains largely unknown. In this study, the inhibition of melanocyte function was observed in C57BL/6J mice modeled through chronic restraint stress. Furthermore, downregulation of the expression of the long noncoding RNAs Mir17hg was identified using RNA sequencing. The regulatory role of Mir17hg in melanogenesis was also investigated in melanocytes and zebrafish embryos through overexpression or knockdown. Finally, TGFß receptor 2 was shown to be a downstream target in Mir17hg-mediated melanogenesis regulation, in which the classical TGFß/SMAD signaling cascade and the PI3K/AKT/mTOR signaling cascade play important roles. In conclusion, our results revealed an important regulatory role of Mir17hg in melanogenesis through inhibition of TGFßR2, which can provide a potential therapeutic target for treating skin depigmentation disorders.

Traditional Chinese Medicine formula, Sanwujiao granule, attenuates ischemic stroke by promoting angiogenesis through early administration.

ETHNOPHARMACOLOGICAL RELEVANCE: Ischemic stroke (IS) is one of the most lethal diseases with the insufficient pharmacology therapeutic approach. Sanwujiao granule (SW) is widely used for IS in China with little known about its underlying mechanism. AIM OF THE STUDY: To investigate the characteristics of therapeutic effects and potential mechanisms of SW against IS. MATERIALS AND METHODS: The fingerprint of SW was applied by high-performance liquid chromatography-mass spectrometry (HPLC-MS). Three different drug treatment strategies, including prophylactic administration, early administration and delayed administration, were applied in rats' permanent middle cerebral occlusion (pMCAO) model. The Garcia neurological deficit test, adhesive removal test, rotarod test, TTC and TUNEL staining were performed to evaluate the pathological changes. The transcriptomic analysis was used to predict the potential mechanism of SW. The vascular deficiency model of Tg(kdrl:eGFP) zebrafish larvae and oxygen-glucose deprivation model on bEnd.3 cells were used to verify SW's pharmacological effect. qRT-PCR, immunofluorescent staining and Western Blot were applied to detect the expression of genes and proteins. The network pharmacology approach was applied to discover the potential bioactive compounds in SW that contribute to its pharmacological effect. RESULTS: SW early and delayed administration attenuated cerebral infarction, neurological deficit and cell apoptosis. The transcriptomic analysis revealed that SW activated angiogenesis-associated biological processes specifically by early administration. CD31 immunofluorescent staining further confirmed the microvessel intensity in peri-infarct regions was significantly elevated after SW early treatment. Additionally, on the vascular deficiency model of zebrafish larvae, SW showed the angiogenesis effect. Next, the cell migration and tube formation were also observed in the bEnd.3 cells with the oxygen-glucose deprivation induced cell injury. It's worth noting that both mRNA and protein levels of angiogenesis factor, insulin-like growth factor 1, were significantly elevated in the pMCAO rats' brains treated with SW. The network pharmacology approach was applied and chasmanine, karacoline, talatisamine, etc. were probably the main active compounds of SW in IS treatment as they affected the angiogenesis-associated targets. CONCLUSIONS: These results demonstrate that SW plays a critical role in anti-IS via promoting angiogenesis through early administration, indicating that SW is a candidate herbal complex for further investigation in treating IS in the clinical.

Optimization of Monobenzone-Induced Vitiligo Mouse Model by the Addition of Chronic Stress.

Vitiligo is a common primary, limited or generalized skin depigmentation disorder. Its pathogenesis is complex, multifactorial and unclear. For this reason, few animal models can simulate the onset of vitiligo, and studies of drug interventions are limited. Studies have found that there may be a pathophysiological connection between mental factors and the development of vitiligo. At present, the construction methods of the vitiligo model mainly include chemical induction and autoimmune induction against melanocytes. Mental factors are not taken into account in existing models. Therefore, in this study, mental inducement was added to the monobenzone (MBEH)-induced vitiligo model. We determined that chronic unpredictable mild stress (CUMS) inhibited the melanogenesis of skin. MBEH inhibited melanin production without affecting the behavioral state of mice, but mice in the MBEH combined with CUMS (MC) group were depressed and demonstrated increased depigmentation of the skin. Further analysis of metabolic differences showed that all three models altered the metabolic profile of the skin. In summary, we successfully constructed a vitiligo mouse model induced by MBEH combined with CUMS, which may be better used in the evaluation and study of vitiligo drugs.

Peripheral benzodiazepine receptor TSPO needs to be reconsidered before using as a drug target for a pigmentary disorder.

The peripheral benzodiazepine receptor (TSPO/PBR) is highly conserved among different species but with perplexing biochemical functions. Multiple ligands of TSPO show commendable regulatory activities in lots of biological functions, such as neuro-protection, cholesterol transport, and so on. These researches support that TSPO may be a potential target for disease treatment and drug development. Previous studies have shown that its ligands benzodiazepines show a satisfactory effect on melanogenic promotion. However, the potential application of TSPO in drug development for pigmentary disorder needs further investigation. In this study, we confirmed the melanogenesis induction of TSPO ligand, Ro5-4864 in mouse melanoma cell lines, human skin tissue, and zebrafish embryos by inducing melanin synthesis and melanosome transport. Molecular genetics and pharmacological studies showed that TSPO deficiency did not affect melanin production in B16F10 cells and zebrafish embryos, nor did it affect the melanin promotion effect of Ro5-4864. Whether or not TSPO exists, the expression of lots of melanogenesis-related proteins, such as TYR, TRP-1, DCT, Mlph, and Rab27 was upregulated with the Ro5-4864 administration. These results indicated that Ro5-4864 induces melanogenesis in a TSPO-independent manner, which is inconsistent with previous research. This research is a reminder that we need to be very careful during target validation in drug development.

Both prokaryotes and eukaryotes produce an immune response against plasmids with 5'-GTTTGTT-3'.

In the evolutionary "arms race" from prokaryotes to eukaryotes, some memories of foreign DNA have been conserved for defensive purposes. Shortly after invasion by the plasmid, pEGFP-N1, the conserved the defense gene, isg15, was activated in the zebrafish zygote and in mammalian cells. Based on the sequence similarity, we found three virus-derived sequences in pEGFP-N1 which share the 5'-GTTTGTT-3' core sequence, an epigenetic factor leading to increased expression of isg15. Mutation of the core sequence greatly reduces the degradation rate of the plasmid in E. coli cells or zebrafish embryos. We conclude that a conserved defense response, common to both eukaryotic and prokaryotic cells, allows identification and degradation of plasmids containing 5'-GTTTGTT-3'.

Serotonin (5-HT) 2A Receptor Involvement in Melanin Synthesis and Transfer via Activating the PKA/CREB Signaling Pathway.

The 5-HT2A serotonin receptor (HTR2A) has been reported to be involved in the serotonin- or serotonin receptor 2A agonist-induced melanogenesis in human melanoma cells. However, the molecular mechanisms underlying HTR2A in regulating melanogenesis remain poorly understood. In this research, cultured mouse melanoma cell line B16F10, human skin, and zebrafish embryos were used to elucidate the downstream signaling of HTR2A in regulating melanogenesis and to verify the potential application of HTR2A in the treatment of pigment-associated cutaneous diseases. We demonstrated that HTR2A antagonists (AT1015 and ketanserin) attenuated the melanogenesis induction of serotonin in both mouse melanoma cells and zebrafish embryos. The agonists of HTR2A (DOI and TCB-2) increased melanin synthesis and transfer in B16F10 cells, human skin tissue, and zebrafish embryos. Furthermore, the HTR2A agonists increased the expression of proteins related to melanosome organization and melanocyte dendrites to facilitate the melanocyte migration and melanosome transport. HTR2A antagonists and genetic knockout of zebrafish htr2aa (the homologue of mammalian HTR2A gene) were also used to clarify that HTR2A mediates serotonin and DOI in regulating melanogenesis. Finally, through small scale screening of the candidate downstream pathway, we demonstrated that HTR2A mediates the melanogenesis induction of its ligands by activating the PKA/CREB signaling pathway. In this research, we further confirmed that HTR2A is a crucial protein to mediate melanocyte function. Meanwhile, this research supports that HTR2A could be designed as a drug target for the development of chemicals to treat cutaneous diseases with melanocytes or melanogenesis abnormality.

Keratinocytes take part in the regulation of substance P in melanogenesis through the HPA axis.

BACKGROUND: Clinical findings have shown that skin depigmentation disorder such as vitiligo may be closely associated with the release of central and peripheral substance P (SP) resulted from chronic psychological stress or sudden mental blow. But the regulatory role of SP and its receptor, tachykinin receptor in the pathogenesis of vitiligo is unclear. OBJECTIVES: To investigate the function and mechanism of SP in melanogenesis. METHODS: The chronic mental stress was used to explore the intrinsic association between psychological factors, SP and melanogenesis disorder. The effect of SP on melanogenesis through hypothalamic pituitary adrenocortical (HPA) axis was studied by skin culture in vitro. The conditioned medium experiment demonstrated the indirect effect of SP on melanogenesis of B16F10 cells through HaCaT cells. The ability to produce melanin was evaluated by detecting melanin and tyrosinase activity. qRT-PCR, western blotting and immunohistochemistry were used to detect the expression of related genes and proteins in melanogenesis and HPA axis. RESULTS: Increased SP expression and reduction of melanogenesis in the skin of mice were observed under mental stress. Melanogenesis was suppressed in the cultured human skin treated with SP due to the down-regulation of melanin-related proteins and HPA axis genes. The melanogenesis of B16F10 cells was inhibited by the conditioned medium of HaCaT cells treated with SP. CONCLUSIONS: Overall, these results indicate that excess SP originated from mental stress interferes with melanogenesis through keratinocytes in the skin. The HPA axis is the key downstream to perceive the SP signaling and furtherly regulate the melanogenesis.

Zebrafish Foxc1a controls ventricular chamber maturation by directly regulating wwtr1 and nkx2.5 expression.

Chamber maturation is a significant process in cardiac development. Disorders of this crucial process lead to a range of congenital heart defects. Foxc1a is a critical transcription factor reported to regulate the specification of cardiac progenitor cells. However, little is known about the role of Foxc1a in modulating chamber maturation. Previously, we reported that foxc1a-null zebrafish embryos exhibit disrupted heart structures and functions. In this study, we observe that ventricle structure and cardiomyocyte proliferation are abolished during chamber maturation in foxc1a-null zebrafish embryos. To observe the endogenous localization of Foxc1a in the hearts of living embryos, we insert eyfp at the foxc1a genomic locus using TALEN. Analysis of the knockin zebrafish show that foxc1a is widely expressed in ventricular cardiomyocytes during chamber development. Cardiac RNA sequencing analysis reveals the downregulated expression of the Hippo signaling effector wwtr1. Dual-luciferase and chromatin immunoprecipitation assays reveal that Foxc1a can bind directly to three sites in the wwtr1 promoter region. Furthermore, wwtr1 mRNA overexpression is sufficient to reverse the ventricle defects during chamber maturation. Conditional overexpression of nkx2.5 also partially rescues the ventricular defects during chamber development. These findings demonstrate that wwtr1 and nkx2.5 are direct targets of Foxc1a during ventricular chamber maturation.

Dendrobium catenatum Lindl. Water Extracts Attenuate Atherosclerosis.

OBJECTIVES: Dendrobium catenatum Lindl. (DH) is a Chinese herbal medicine, which is often used to make tea to improve immunity in China. Rumor has it that DH has a protective effect against cardiovascular disease. However, it is not clear how DH can prevent cardiovascular disease, such as atherosclerosis (AS). Therefore, the purpose of this study is to study whether DH can prevent AS and the underlying mechanisms. METHODS: Zebrafish larvae were fed with high-cholesterol diet (HCD) to establish a zebrafish AS model. Then, we used DH water extracts (DHWE) to pretreat AS zebrafish. The plaque formation was detected by HE, EVG, and oil red O staining. Neutrophil and macrophage counts were calculated to evaluate the inflammation level. Reactive oxygen species (ROS) activity, malondialdehyde (MDA) content, and superoxide dismutase (SOD) activity in zebrafish were measured to reflect oxidative stress. The cholesterol accumulation and the levels of lipid, triglyceride (TG), and total cholesterol (TC) were measured to reflect lipid metabolism disorder. Then, parallel flow chamber was utilized to establish a low shear stress- (LSS-) induced endothelial cell (EC) dysfunction model. EA.hy926 cells were exposed to LSS (3 dyn/cm2) for 30 min and treated with DHWE. The levels of ROS, SOD, MDA, glutathione (GSH), and glutathiol (GSSG) in EA.hy926 cells were analysed to determine oxidative stress. The release of nitric oxide (NO), endothelin-1 (ET-1), and epoprostenol (PGI2) in EA.hy926 cells was measured to reflect EC dysfunction. The mRNA expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in EA.hy926 cells was detected to reflect EC dysfunction inflammation. RESULTS: The results showed that DHWE significantly reduced cholesterol accumulation and macrophage infiltration in early AS. Finally, DHWE significantly alleviate the lipid metabolism disorder, oxidative stress, and inflammation to reduce the plaque formation of AS zebrafish larval model. Meanwhile, we also found that DHWE significantly improved LSS-induced EC dysfunction and oxidative stress in vitro. CONCLUSION: Our results indicate that DHWE could be used as a prevention method to prevent AS.

Zebrafish Model for Screening Antiatherosclerosis Drugs.

This study is aimed at establishing a zebrafish model of AS, which can be applied for high-throughput screening anti-AS drugs. A zebrafish AS model was induced by high cholesterol diet (HCD) and lipopolysaccharide (LPS). In the early stage of modeling, HCD induced zebrafish to show some early symptoms similar to human AS, mainly cholesterol accumulation, vascular inflammation, lipid metabolism disorder, and oxidative stress. In addition to lipid metabolism disorders, LPS also induced the same symptoms. And when HCD and LPS exist at the same time, these AS symptoms in zebrafish become more severe. When the modeling time reached 45 days, HCD and LPS induce the formation of plaques in zebrafish blood vessels, and these plaques contain fibrous tissue and lipids, which are similar to human AS plaques. We also evaluated the efficacy of some anti-AS drugs (atorvastatin, aspirin, and vitamin C) through these zebrafish AS models. The results found that atorvastatin can significantly reduce the symptoms of AS induced by HCD and LPS, and aspirin and vitamins can significantly reduce the symptoms of AS induced by LPS. It is feasible to use zebrafish to establish an AS model, and the zebrafish AS model can be used for high-throughput screening of anti-AS drugs.

The G4 resolvase RHAU modulates mRNA translation and stability to sustain postnatal heart function and regeneration.

Post-transcriptional regulation of mRNA translation and stability is primarily achieved by RNA-binding proteins, which are of increasing importance for heart function. Furthermore, G-quadruplex (G4) and G4 resolvase activity are involved in a variety of biological processes. However, the role of G4 resolvase activity in heart function remains unknown. The present study aims to investigate the role of RNA helicase associated with adenylate- and uridylate-rich element (RHAU), an RNA-binding protein with G4 resolvase activity in postnatal heart function through deletion of Rhau in the cardiomyocytes of postnatal mice. RHAU-deficient mice displayed progressive pathological remodeling leading to heart failure and mortality and impaired neonatal heart regeneration. RHAU ablation reduced the protein levels but enhanced mRNA levels of Yap1 and Hexim1 that are important regulators for heart development and postnatal heart function. Furthermore, RHAU was found to associate with both the 5' and 3' UTRs of these genes to destabilize mRNA and enhance translation. Thus, we have demonstrated the important functions of RHAU in the dual regulation of mRNA translation and stability, which is vital for heart physiology.

5-Hydroxytryptamine (5-HT) Positively Regulates Pigmentation via Inducing Melanoblast Specification and Melanin Synthesis in Zebrafish Embryos.

It has been reported that 5-hydroxytryptamine (5-HT) is related to melanogenesis in mice and melanoma cells. However, the underlying mechanisms of 5-HT in regulating pigmentation remains unknown. In this study, we aim to clarify the regulatory mechanism of 5-HT in the pigmentation of zebrafish embryos and B16F10 cells. Our results show that 5-HT induces the pigmentation of zebrafish embryos in a dosage-dependent manner at concentrations of 0.01-1 mM. Whole mount in situ hybridizations and qRT-PCR in zebrafish embryos indicate that the expression of neural crest cells marker gene sox10 is not changed in embryos treated with 5-HT compared to control group. The expression of mitfa, the marker gene of melanoblasts, is increased in the presence of 5-HT. Furthermore, 5-HT increased the expression of regeneration associated genes, namely kita, mitfa, and dct, after ablation of the melanogenic cells in zebrafish embryos. The experiments in B16F10 cells show that 5-HT promotes melanin synthesis by up-regulating the expression of key proteins MITF, TYR, TRP-1, and TRP-2. Especially, the small molecule inhibitor of PKA signaling, but not AKT and MAPK signaling, attenuates the up-regulation of MITF and TYR resulted from 5-HT induction in B16F10 cells. These results will help us to further understand the regulatory network of vertebrate pigmentation.

Axenfeld-Rieger syndrome-associated mutants of the transcription factor FOXC1 abnormally regulate NKX2-5 in model zebrafish embryos.

FOXC1 is a member of the forkhead family of transcription factors, and whose function is poorly understood. A variety of FOXC1 mutants have been identified in patients diagnosed with the autosomal dominant disease Axenfeld-Rieger syndrome, which is mainly characterized by abnormal development of the eyes, particularly those who also have accompanying congenital heart defects (CHD). However, the role of FOXC1 in CHD, and how these mutations might impact FOXC1 function, remains elusive. Our previous work provided one clue to possible function, demonstrating that zebrafish foxc1a, an orthologue of human FOXC1 essential for heart development, directly regulates the expression of nkx2.5, encoding a transcriptional regulator of cardiac progenitor cells. Abnormal expression of Nkx2-5 leads to CHD in mice and is also associated with CHD patients. Whether this link extends to the human system, however, requires investigation. In this study, we demonstrate that FOXC1 does regulate human NKX2-5 expression in a dose-dependent manner via direct binding to its proximal promoter. A comparison of FOXC1 mutant function in the rat cardiac cell line H9c2 and zebrafish embryos suggested that the zebrafish embryos might serve as a more representative model system than the H9c2 cells. Finally, we noted that three of the Axenfeld-Rieger syndrome FOXC1 mutations tested increased, whereas a fourth repressed the expression of NKX2-5 These results imply that mutant FOXC1s might play etiological roles in CHD by abnormally regulating NKX2-5 in the patients. And zebrafish embryos can serve as a useful in vivo platform for rapidly evaluating disease-causing roles of mutated genes.

Protective Activities of Dendrobium huoshanense C. Z. Tang et S. J. Cheng Polysaccharide against High-Cholesterol Diet-Induced Atherosclerosis in Zebrafish.

Cardiovascular disease is the highest cause of death, and atherosclerosis (AS) is the primary pathogenesis of many cardiovascular diseases. In this study, we aim to investigate the possible pharmaceutical effects of Dendrobium huoshanense C. Z. Tang et S. J. Cheng polysaccharide (DHP) in AS. We fed zebrafish with high-cholesterol diet (HCD) to establish a zebrafish AS model and treated with DHP and observed plaque formation and neutrophil counts under a fluorescence microscope. Next, a parallel flow chamber was utilized to establish low shear stress- (LSS-) induced endothelial cell (EC) dysfunction model. We observed that DHP significantly improved HCD-induced lipid deposition, oxidative stress, and inflammatory response, mainly showing that DHP significantly increased superoxide dismutase (SOD) activity, decreased plaque formation, and decreased neutrophil recruitment and the levels of total cholesterol (TC), triglyceride (TG), malondialdehyde (MDA), and reactive oxygen species (ROS). Furthermore, DHP significantly improved LSS-induced oxidative stress and EC dysfunction. Our results indicated that DHP can exert treatment effects on AS, which may attribute to its hypolipidemic, antioxidant, anti-inflammatory activities and improving LSS-induced EC dysfunction. DHP has promising potential for further development as a functional natural medicine source targeted at AS prevention.

Zebrafish microRNA miR-210-5p inhibits primitive myelopoiesis by silencing foxj1b and slc3a2a mRNAs downstream of gata4/5/6 transcription factor genes.

Zebrafish gata4/5/6 genes encode transcription factors that lie on the apex of the regulatory hierarchy in primitive myelopoiesis. However, little is known about the roles of microRNAs in gata4/5/6-regulated processes. Performing RNA-Seq deep sequencing analysis of the expression changes of microRNAs in gata4/5/6-knockdown embryos, we identified miR-210-5p as a regulator of zebrafish primitive myelopoiesis. Knocking down gata4/5/6 (generating gata5/6 morphants) significantly increased miR-210-5p expression, whereas gata4/5/6 overexpression greatly reduced its expression. Consistent with inhibited primitive myelopoiesis in the gata5/6 morphants, miR-210-5p overexpression repressed primitive myelopoiesis, indicated by reduced numbers of granulocytes and macrophages. Moreover, knocking out miR-210 partially rescued the defective primitive myelopoiesis in zebrafish gata4/5/6-knockdown embryos. Furthermore, we show that the restrictive role of miR-210-5p in zebrafish primitive myelopoiesis is due to impaired differentiation of hemangioblast into myeloid progenitor cells. By comparing the set of genes with reduced expression levels in the gata5/6 morphants to the predicted target genes of miR-210-5p, we found that foxj1b and slc3a2a, encoding a forkhead box transcription factor and a solute carrier family 3 protein, respectively, are two direct downstream targets of miR-210-5p that mediate its inhibitory roles in zebrafish primitive myelopoiesis. In summary, our results reveal that miR-210-5p has an important role in the genetic network controlling zebrafish primitive myelopoiesis.

The transcription factor Foxc1a in zebrafish directly regulates expression of nkx2.5, encoding a transcriptional regulator of cardiac progenitor cells.

Cardiogenesis is a tightly controlled biological process required for formation of a functional heart. The transcription factor Foxc1 not only plays a crucial role in outflow tract development in mice, but is also involved in cardiac structure formation and normal function in humans. However, the molecular mechanisms by which Foxc1 controls cardiac development remain poorly understood. Previously, we reported that zebrafish embryos deficient in foxc1a, an ortholog of mammalian Foxc1, display pericardial edemas and die 9-10 days postfertilization. To further investigate Foxc1a's role in zebrafish cardiogenesis and identify its downstream target genes during early heart development, we comprehensively analyzed the cardiovascular phenotype of foxc1a-null zebrafish embryos. Our results confirmed that foxc1a-null mutants exhibit disrupted cardiac morphology, structure, and function. Performing transcriptome analysis on the foxc1a mutants, we found that the expression of the cardiac progenitor marker gene nkx2.5 was significantly decreased, but the expression of germ layer-patterning genes was unaffected. Dual-fluorescence in situ hybridization assays revealed that foxc1a and nkx2.5 are co-expressed in the anterior lateral plate mesoderm at the somite stage. Chromatin immunoprecipitation and promoter truncation assays disclosed that Foxc1a regulates nkx2.5 expression via direct binding to two noncanonical binding sites in the proximal nkx2.5 promoter. Moreover, functional rescue experiments revealed that developmental stage-specific nkx2.5 overexpression partially rescues the cardiac defects of the foxc1a-null embryos. Taken together, our results indicate that during zebrafish cardiogenesis, Foxc1a is active directly upstream of nkx2.5.

Zebrafish Znfl1 proteins control the expression of hoxb1b gene in the posterior neuroectoderm by acting upstream of pou5f3 and sall4 genes.

Transcription factors play crucial roles in patterning posterior neuroectoderm. Previously, zinc finger transcription factor znfl1 was reported to be expressed in the posterior neuroectoderm of zebrafish embryos. However, its roles remain unknown. Here, we report that there are 13 copies of znfl1 in the zebrafish genome, and all the paralogues share highly identical protein sequences and cDNA sequences. When znfl1s are knocked down using a morpholino to inhibit their translation or dCas9-Eve to inhibit their transcription, the zebrafish gastrula displays reduced expression of hoxb1b, the marker gene for the posterior neuroectoderm. Further analyses reveal that diminishing znfl1s produces the decreased expressions of pou5f3, whereas overexpression of pou5f3 effectively rescues the reduced expression of hoxb1b in the posterior neuroectoderm. Additionally, knocking down znfl1s causes the reduced expression of sall4, a direct regulator of pou5f3, in the posterior neuroectoderm, and overexpression of sall4 rescues the expression of pou5f3 in the knockdown embryos. In contrast, knocking down either pou5f3 or sall4 does not affect the expressions of znfl1s Taken together, our results demonstrate that zebrafish znfl1s control the expression of hoxb1b in the posterior neuroectoderm by acting upstream of pou5f3 and sall4.

An alternative novel tool for DNA editing without target sequence limitation: the structure-guided nuclease.

Engineered endonucleases are a powerful tool for editing DNA. However, sequence preferences may limit their application. We engineer a structure-guided endonuclease (SGN) composed of flap endonuclease-1 (FEN-1), which recognizes the 3' flap structure, and the cleavage domain of Fok I (Fn1), which cleaves DNA strands. The SGN recognizes the target DNA on the basis of the 3' flap structure formed between the target and the guide DNA (gDNA) and cut the target through its Fn1 dimerization. Our results show that the SGN, guided by a pair of gDNAs, cleaves transgenic reporter gene and endogenous genes in zebrafish embryonic genome.

Retinoic Acid Signaling Is Essential for Valvulogenesis by Affecting Endocardial Cushions Formation in Zebrafish Embryos.

Retinoic acid (RA) plays important roles in many stages of heart morphogenesis. Zebrafish embryos treated with exogenous RA display defective atrio-ventricular canal (AVC) specification. However, whether endogenous RA signaling takes part in cardiac valve formation remains unknown. Herein, we investigated the role of RA signaling in cardiac valve development by knocking down aldh1a2, the gene encoding an enzyme that is mainly responsible for RA synthesis during early development, in zebrafish embryos. The results showed that partially knocking down aldh1a2 caused defective formation of primitive cardiac valve leaflets at 108 hpf (hour post-fertilization). Inhibiting endogenous RA signaling by 4-diethylaminobenzal-dehyde revealed that 16-26 hpf was a key time window when RA signaling affects the valvulogenesis. The aldh1a2 morphants had defective formation of endocardial cushion (EC) at 76 hpf though they had almost normal hemodynamics and cardiac chamber specification at early development. Examining the expression patterns of AVC marker genes including bmp4, bmp2b, nppa, notch1b, and has2, we found the morphants displayed abnormal development of endocardial AVC but almost normal development of myocardial AVC at 50 hpf. Being consistent with the reduced expression of notch1b in endocardial AVC, the VE-cadherin gene cdh5, the downstream gene of Notch signaling, was ectopically expressed in AVC of aldh1a2 morphants at 50 hpf, and overexpression of cdh5 greatly affected the formation of EC in the embryos at 76 hpf. Taken together, our results suggest that RA signaling plays essential roles in zebrafish cardiac valvulogenesis.

Zebrafish foxc1a plays a crucial role in early somitogenesis by restricting the expression of aldh1a2 directly.

Foxc1a is a member of the forkhead transcription factors. It plays an essential role in zebrafish somitogenesis. However, little is known about the molecular mechanisms underlying its controlling somitogenesis. To uncover how foxc1a regulates zebrafish somitogenesis, we generated foxc1a knock-out zebrafish using TALEN (transcription activator-like effector nuclease) technology. The foxc1a null embryos exhibited defective somites at early development. Analyses on the expressions of the key genes that control processes of somitogenesis revealed that foxc1a controlled early somitogenesis by regulating the expression of myod1. In the somites of foxc1a knock-out embryos, expressions of fgf8a and deltaC were abolished, whereas the expression of aldh1a2 (responsible for providing retinoic acid signaling) was significantly increased. Once the increased retinoic acid level in the foxc1a null embryos was reduced by knocking down aldh1a2, the reduced expression of myod1 was partially rescued by resuming expressions of fgf8a and deltaC in the somites of the mutant embryos. Moreover, a chromatin immunoprecipitation assay on zebrafish embryos revealed that Foxc1a bound aldh1a2 promoter directly. On the other hand, neither knocking down fgf8a nor inhibiting Notch signaling affected the expression of aldh1a2, although knocking down fgf8a reduced expression of deltaC in the somites of zebrafish embryos at early somitogenesis and vice versa. Taken together, our results demonstrate that foxc1a plays an essential role in early somitogenesis by controlling Fgf and Notch signaling through restricting the expression of aldh1a2 in paraxial mesoderm directly.